Transcript CBP
Protein Purification Involving a Unique Auto-Cleavage Feature of a Repeated EAAAK Peptide Interdisciplinary Research of Enzyme Technology Protein Production Structure & Catalytic Mechanism Bio-conversion Enzyme Research Bio-medical application Interdisciplinary Research of Enzyme Technology Expression systems Purification Techniques Protein Production Structure & Catalytic Glycoside hydrolases GH-1,Mechanism Bio-conversion Enzyme Research Oligosaccharide preparation Biomass degradation Transglycosylation GH-3, GH-17, GH-18, GH-29, GH-46, GH-54, GH-64, GH-72, GH-75 Bio-medical application LC/MS/MS Biosensor/SPR SiNW-FET Nano-particles Purification Animal tissue Plant materials Grinding Extraction Fermentation Extracellular enzymes Filtration Concentration Microorganisms Purification Intracellular enzymes Disruption Pure Enzyme Strategy for massive production and purification of protein Current strategies and problems Recombinant protein technology is the best solution so far. Current strategies and problems To simplify purification of recombinant proteins, several engineered affinity tags are used with which fusion protein can be purified to near homogeneity in a simple procedure. Linker Carrier Protein Target protein Protein purification based on affinity binding Linker Affinity matrix Carrier Protein Target protein Binding Protein purification based on affinity binding Binding Affinity matrix Linker Carrier Protein Target protein • Glutathione S-transferase (Novagen, GST) • Maltose-binding protein (pMAL system, NEB) • Chitin-binding domain (IMPACT system, NEB) Protein purification based on affinity binding excess wash Dialysis to remove Current strategies Carrier protein (or Tag) may need to be removed, commonly by protease, after fusion protein has been purified before subsequent use in downstream application. Common Drawbacks Linker Affinity matrix • Costly affinity matrix required. • Post proteolytic process needed. Protein purification based on affinity binding Binding Affinity matrix Linker Carrier Protein Target protein • Glutathione S-transferase (Novagen, GST) • Maltose-binding protein (pMAL system, NEB) • Chitin-binding domain (IMPACT system, NEB) • Chitin-binding protein with auto-cleavage peptide linker (developed by NCTU) A new system developed by our group A vector containing chitin-binding protein and repeated EAAAK peptide linker to form a simple and cost-effective system for protein expression and purification. CBP Linker Repeated EAAAK peptide with auto-cleavage property MCS History of our finding…… Starting from the study of Chitinase from Bacillus NCTU2 Characteristics of chitin-binding Protein (CBP) • CBP promotes the hydrolysis of chitin catalyzed by chitinase. • CBP has good binding specificity for chitin. • pH＞8, CBP can bind to chitin. • pH＜6, CBP can be eluted. Structures of Chitinases TIM-barrel structure Serratia ChiA Bacillus NCTU2 ChiA Catalytic active ? Linker Chitin-binding domain (1~150 aa) Vector design Linker amino acid sequence 1 2 3 4 5 6 7 AKRGWI GTGGEGPGG GTGGEGPGGGGPGEGGTG TTLGSNLGTLGLK (EAAAK)5 GTGGEGPGGGGPGEGGTGGTGGEGPGG (GGGGS)5 Nde1 PT7 CBP21 gene Linker 1~7 protease cutting site A Procedure of pRSET/CBP-V5G vector construction CBP Linker Protease cutting site MCS The chimeric chitinase is active without significant improvement in catalysis. However, interestingly…… Linker The fusion protein broke into two fragments at pH 6.0! The fusion protein broke into two fragments at pH 6.0! CBP-V5G-ChiA (MW 58 kDa) NCTU2 ChiA (36 kDa) CBP (19 kDa) Lane 1：Sample kept in water for hours (pH 6.9). Lane 2：Sample in Pi buffer (50 mM, pH 6.0) for 1 day Lane 3：Sample in Pi buffer (50, mM, pH 6.0) for 2 days Lane 4：Sample kept in water (pH 6.9) for 1 day Lane 5：Sample kept in water (pH 6.9) for 2 day Other cases CBP-V5G-CNS( 45 kDa) CNS (chitosanase, 24kDa) 22.5 CBP (19 kDa) Exchange buffers with pH 4.2 8.0 and kept at 25 ℃ for 12 h. Dose Auto-cleavage occur on CBP-(EAAAK)5-G-ChiA? Or contamination of protease? M pH8.0 7.5 7.0 6.0 5.0 4.2 66.2 45 CBP-V5G-CNS ( 45kDa) 35 22.5 18.4 100 ℃ for 10 min under pH 3.6； exchange buffers with pH 4.2 8.0 and then kept at 25 ℃ for 12 h. 14 pH-dependent auto-cleavage of (EAAAK)5 linker!! Construction of fusion CNS with various repeated EAAAK linkers • CBP- (EAAAK)2 G-CNS • CBP- (EAAAK)3 G-CNS • CBP- (EAAAK)4 G-CNS • CBP- (EAAAK)5 G-CNS • CBP- (EAAAK)5 -CNS (Fusion protein without genenase I cutting site) • (EAAAK)5 G-CNS (Fusion protein without CBP) The fusion proteins were incubated in phosphate buffer (pH 6.0 at 16 ℃) so that partial auto-cleavage fragments can be obtained. SDS PAGE and MS analyses of auto-cleavage of the fusion Protein Lane 1: protein marker Lane 2: CBP-V2G-CNS Lane 3: CBP-V3G-CNS Lane 4: CBP-V4G-CNS Lane 5: CBP-V5G-CNS Lane 6: CBP-V5-CNS Lane 7: V5G-CNS SDS PAGE and MS analyses of autocleavage of the fusion Protein Lane 1: protein marker Lane 2: CBP-V2G-CNS Lane 3: CBP-V3G-CNS Lane 4: CBP-V4G-CNS Lane 5: CBP-V5G-CNS Lane 6: CBP-V5-CNS Lane 7: V5G-CNS SDS PAGE and MS analyses of autocleavage of the fusion Protein Lane 1: protein marker Lane 2: CBP-V2G-CNS Lane 3: CBP-V3G-CNS Lane 4: CBP-V4G-CNS Lane 5: CBP-V5G-CNS Lane 6: CBP-V5-CNS Lane 7: V5G-CNS SDS PAGE and MS analyses of autocleavage of the fusion Protein Lane 1: protein marker Lane 2: CBP-V2G-CNS Lane 3: CBP-V3G-CNS Lane 4: CBP-V4G-CNS Lane 5: CBP-V5G-CNS Lane 6: CBP-V5-CNS Lane 7: V5G-CNS SDS PAGE and MS analyses of autocleavage of the fusion Protein Lane 1: protein marker Lane 2: CBP-V2G-CNS Lane 3: CBP-V3G-CNS Lane 4: CBP-V4G-CNS Lane 5: CBP-V5G-CNS Lane 6: CBP-V5-CNS Lane 7: V5G-CNS SDS PAGE and MS analyses of autocleavage of the fusion Protein Lane 1: protein marker Lane 2: CBP-V2G-CNS Lane 3: CBP-V3G-CNS Lane 4: CBP-V4G-CNS Lane 5: CBP-V5G-CNS Lane 6: CBP-V5-CNS Lane 7: V5G-CNS Protocol of one-pot protein purification CBP-V5G-CNS CBP-V5G-LPHase Lane 1: marker Lane 2: crude enzyme Lane 3: β-chitin purified enzyme Lane 4: After auto-cleavage, the obtained target protein CNS: 24 kDa, LPHase: 40 kDa Purification of His-Tagged Recombinant protein using Nickel column His-Tagged protein can bind to nickel column with moderate affinity and can be eluted with high concentration of imidazole. His-Tag + auto-cleavage peptide + magnetic particles Will it work?? One-step protein purification using MP and ACP T a rge t pro tein N o n -tag g e d pro tein 12 0 0 0 rp m u ltra -so n icatio n 1 2 00 0 rp m crud e p rote in H is-tag b in din g W ash & cle a vage re m o v e M P His8-GFP-(EAAAK)2-mcherry 1 75kD 63kD 48kD 35kD 28kD 17kD 10kD 2 3 4 Lane 1: marker Lane 2: crude enzyme Lane 3: bound protein Lane 4: unbound protein after autocleavage His6-(EAAAK)3-GFP 1 2 3 4 100kD 75kD 63kD 48kD 35kD 28kD 17kD 10kD Lane 1: marker Lane 2: crude enzyme Lane 3: MP bound with protein Lane 4: unbound protein after autocleavage Conclusions • The repeated EAAAK peptide exhibited an auto-cleavage feature which can be mediated by pH condition. • With this system, many proteins have been successfully purified. • Integration of auto-cleavage peptide (ACP) technique with NTA-coated magnetic particles coated can simplify the purification process.